Evidence of increased synthesis of δ-aminolevulinic acid dehydratase in experimental lead-poisoned rats

δ-Aminolevulinic acid dehydratase (porphobilinogen synthase; 5-aminolevulinate hydro-lyase, EC 4.2.1.24) was purified from rat and rabbit erythrocytes to a homogeneous state. Specific activities were 26.0 and 26.6 units/mg protein for the rat and rabbit enzymes, respectively, and their estimated molecular weight was 280 000, each consisting of 8 subunits of M_r 35 000. In order to quantitate rat δ-aminolevulinic acid dehydratase at several stages of lead-poisoning, a radioimmunoassay technique using goat antiserum against the rat enzyme was developed for the first time. This technique was specific, reproducible and high sensitive allowing determination of 1 ng enzyme. When drinking water containing 25 mM lead acetate was given daily to rats ad lib. the δ-aminolevulinic acid dehydratase activity in the blood, assayed without any pretreatment, decreased to 8% of the control level on the next day. On the contrary, the restored enzyme activity, assayed in the presence of Zn²⁺ and dithiothreitol, was greater than normal by the fourth day of lead administration in bone-marrow cells and by the ninth day in the peripheral blood. The increased activity level stayed the same from the ninth day onward. The enzyme content as determined directly by the radioimmunoassay technique at this stage was about 2-fold above that the control. There was no significant difference in the number of reticulocytes and the distribution profile of different types of reticulocytes between the lead-exposed and non-exposed rats. Therefore, the increase in the amount of δ-aminolevulinic acid dehydratase in erythrocytes of lead-poisoned rats was suggested to be due to an increased rate of synthesis in the bone-marrow cells.     

Light and electron microscopic studies on experimental nocardia-toxicosis in mice

An exotoxin (HS-6) produced by Nocardia otitidiscaviarum isolated from certain lesions of cutaneous nocardiosis of a male 82-year-old patient induced severe injuries in the pancreas, liver, stomach, small intestine, heart, thymus and kidney of male ICR mice. Mice given Nocardia-free preparation of HS-6 at a dose of 1 mg/kg of body weight developed several autophagic vacuoles in the pancreas and liver within 20 min after the i.p. injection. Thereafter, the autophagic vacuoles increased in number and size with time. About 24 hr after the administration of HS-6, the liver showed marked accumulation of fat droplets in the cytoplasm of the hepatocytes. Although they contained abundant autophagic vacuoles in the regions of RER, there were no lipomatoses in the acinar cells of the pancreas, those of the chief cells and smooth muscle cells of the stomach, Paneth cells, goblet cells, smooth muscle cells of the small intestine, and plasma cells in the digestive tract. Biochemical examinations revealed that HS-6 had no significant effect on the protein synthesis of reticulocytes. Inoculation of the Nocardia into the mouse peritoneal cavities caused marked granulomatoses in the pancreas, liver and regional lymph nodes, but did not develop autophagic vacuoles in RER regions of these organs.     

Occurrence of Prorocentrum lima,a DSP toxin-producing species from the Atlantic coast of Canada

A species of Prorocentrum (Dinophyta, Prorocentrales), isolated from a phytoplankton net sample from the Atlantic coast of Nova Scotia, has been brought into unialgal culture. The sample was collected at an aquaculture site immediately following an incident of diarrhetic shellfish poisoning (DSP) due to the consumption of contaminated mussels. This clonal isolate has been identified as P. lima, based on its morphological characteristics. Analysis of the culture extract, using high performance liquid chromatography (HPLC) with fluorescence detection, indicated the presence of the DSP toxins, okadaic acid (OA) and dinophysistoxin-1 (DTX-1).     

LSU rDNA-BASED RFLP ASSAYS FOR DISCRIMINATING SPECIES AND STRAINS OF ALEXANDRIUM (DINOPHYCEAE)1

In a previous study large-subunit ribosomal RNA gene (LSU rDNA) sequences from the marine dinoflagellates Alexandrium tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense Balech, A. affine (Fukuyo et Inoue) Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech were compared to assess inter- and intraspecific relationships. Many cultures compared in that study contained more than one class of LSU rDNA. Sequencing pooled clones of rDNA from single cultures revealed length heterogeneities and sequence ambiguities. This complicated sequence comparisons because multiple rDNA clones from a single culture had to be sequenced individually to document the different classes of molecules present in that culture. A further complication remained as to whether or not the observed intraculture sequence variations were reliable genetic markers or were instead artifacts of the polymerase chain reaction (PCR) amplification, cloning, and/or sequencing methods employed. The goals of the present study were to test the accuracy of Alexandrium LSU rDNA sequences using restriction fragment-length polymorphism (RFLP) analysis and to devise RFLP-based assays for discriminating among representatives of that group. Computer-assisted examination of the sequences allowed us to identify a set of restriction enzymes that were predicted to reveal species, strain, and intraculture LSU rDNA heterogeneities. All groups identified by sequencing were revealed independently and repeatedly by RFLP analysis of PCR-amplified material. Five ambiguities and one length heterogeneity, each of which ascribes a unique group of Alexandrium species or strains, were confirmed by restriction digests. Observed intraculture LSU rDNA heterogeneities were not artifacts of cloning and sequencing but were instead a good representation of the spectrum of molecules amplified during PCR reactions. Intraculture LSU rDNA heterogeneities thus serve as unique genetic markers for particular strains of Alexandrium, particularly those of A. tamarense, A. catenella, and A. fundyense. However, some of these “signature heterogeneities” represented a smaller portion of PCR product than was expected given acquired sequences. Other deviations from predicted RFLP patterns included incomplete digestions and appearance of spurious products. These observations indicate that the diversity of sequences in PCR product pools were greater than that observed by cloning and sequencing. The RFLP tests described here are useful tools for characterizing Alexandrium LSU rDNA to define the evolutionary lineage of cultures and are applicable at a fraction of the time, cost, and labor required for sequencing.     

Multiple fatal mycetism caused byAmanita virosa in Mexico

Mushroom poisonings caused by amatoxins are mostly lethal. Information about mycetisms caused by white species ofAmanita is scarce. The present paper describes a case of mushroom poisoning caused byA. virosa. A prolongated latency period (6–10 hours), followed by cholera-like, improvement and visceral complication phases confirmed the amatoxin poisoning. The consumption of about 3 pounds of the toadstool by seven persons caused the death of five. Two patients survive the ingestion.     

Effect of the endoparasite Amoebophrya sp. on toxin content and composition in the paralytic shellfish poisoning dinoflagellate Alexandrium fundyense (Dinophyceae)

Members of the Amoebophrya ceratii complex are endoparasitic dinoflagellates that parasitize a number of their dinoflagellate relatives, including toxic and/or harmful algal bloom-forming species. Despite many studies on the occurrence, prevalence, biology and molecular phylogeny of Amoebophrya spp., little attention has been given to toxin dynamics of host population following parasitism. Using Amoebophrya sp. infecting the paralytic shellfish toxin (PSP)-producing dinoflagellate Alexandrium fundyense, we addressed the following questions: (1) does parasitism by Amoebophrya sp. alter toxin content and toxin profiles of the dinoflagellate A. fundyense over the infection cycle? and (2) do parasite dinospores produced at the end of the infection cycle retain host toxins and thus potentially act as a vector to convey PSP toxin through the marine microbial food-web? Toxin time-course experiments showed that the PSP toxin contents did not vary significantly over the infection cycle, but mean toxin content for infected cultures was significantly higher than that for uninfected cultures. Host toxins were not detected in the free-living, dinospore stage of the parasite. Therefore, our results indicate that Amoebophrya sp. does not function as a vector for transferring PSP toxins to higher trophic levels. Rather, Amoebophrya infections appear to play an important role in maintaining healthy ecosystems by transforming potent toxins-producing dinoflagellates into non-toxic dinospores, representing “edible food” for consumers of the marine microbial food-web during toxic algal bloom event.     

基于Taqman-MGB探针的亚稀褶红菇实时荧光定量PCR检测方法

本研究建立了一种基于Taqman-MGB探针的亚稀褶红菇Russula subnigricans实时荧光定量PCR检测方法。根据亚稀褶红菇与其近似种的内转录间隔区（internal transcribed spacers,ITS）序列差异,设计合成1对引物和1条特异性Taqman-MGB探针,并用常见有毒红菇种类进行验证。结果显示,引物特异性良好,仅亚稀褶红菇出现荧光信号,完成整个检测过程只需2h。该法能够为毒蘑菇中毒的快速检测提供技术支持。     

Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli

Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1–14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1–14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1–14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1–14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis.     

Characterization of Dinophysis spp. (Dinophyceae,Dinophysiales) from the mid-Atlantic region of the United States1

Due to the increasing prevalence of Dinophysis spp. and their toxins on every US coast in recent years, the need to identify and monitor for problematic Dinophysis populations has become apparent. Here, we present morphological analyses, using light and scanning electron microscopy, and rDNA sequence analysis, using a ~2-kb sequence of ribosomal ITS1, 5.8S, ITS2, and LSU DNA, of Dinophysis collected in mid-Atlantic estuarine and coastal waters from Virginia to New Jersey to better characterize local populations. In addition, we analyzed for diarrhetic shellfish poisoning (DSP) toxins in water and shellfish samples collected during blooms using liquid-chromatography tandem mass spectrometry and an in vitro protein phosphatase inhibition assay and compared this data to a toxin profile generated from a mid-Atlantic Dinophysis culture. Three distinct morphospecies were documented in mid-Atlantic surface waters: D. acuminata, D. norvegica, and a “small Dinophysis sp.” that was morphologically distinct based on multivariate analysis of morphometric data but was genetically consistent with D. acuminata. While mid-Atlantic D. acuminata could not be distinguished from the other species in the D. acuminata-complex (D. ovum from the Gulf of Mexico and D. sacculus from the western Mediterranean Sea) using the molecular markers chosen, it could be distinguished based on morphometrics. Okadaic acid, dinophysistoxin 1, and pectenotoxin 2 were found in filtered water and shellfish samples during Dinophysis blooms in the mid-Atlantic region, as well as in a locally isolated D. acuminata culture. However, DSP toxins exceeded regulatory guidance concentrations only a few times during the study period and only in noncommercial shellfish samples.